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Cross-Linking of a DOPA-Containing Peptide Ligand into its G Protein-Coupled Receptor

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2009-04-01
Umanah, George E.
Son, Çağdaş Devrim
Ding, FaXiang
Naider, Fred
Becker, Jeffrey M.
The interaction between a 3,4-dihydroxylphenylalanine (DOPA) labeled analog of the tridecapeptide α-factor (W-H-W-L-Q-L-K-P-G-Q-P-M-Y) and Ste2p, a Saccharomyces cerevisiae model G protein-coupled receptor (GPCR), has been analyzed by periodate-mediated cross-linking. Chemically synthesized α-factor with DOPA substituting for tyrosine at position 13 and biotin tagged onto lysine7 ([Lys7 (BioACA),-Nle12,DOPA13]α-factor; Bio-DOPA-α-factor) was used for crosslinking into Ste2p. The biological activity of Bio-DOPA-α-factor was about one-third that of native α-factor as determined by growth arrest assay and exhibited about a ten-fold lower binding affinity to Ste2p. Bio-DOPA-α-factor cross-linked into Ste2p as demonstrated by western blot analysis using a Neutravidin-HRP conjugate to detect Bio-DOPA-α-factor. Cross-linking was inhibited by excess native α-factor and an α-factor antagonist. The Ste2p-ligand complex was purified using a metal ion affinity column, and after cyanogen bromide treatment, avidin affinity purification was used to capture Bio-DOPA-α-factor-Ste2p cross-linked peptides. MALDI-TOF spectrometric analyses of the cross-linked fragments showed that Bio-DOPA-α-factor reacted with the Phe55-Met69 region of Ste2p. Cross-linking of Bio-DOPA-α-factor was reduced by 80% using a cysteine-less Ste2p (Cys59Ser). These results suggest an interaction between position thirteen of α-factor and residue Cys59 of Ste2p. This study is the first to report DOPA cross-linking of a peptide hormone to a GPCR and the first to identify a residue-to-residue cross-link between Ste2p and α-factor thereby defining a specific contact point between the bound ligand and its receptor.